Geographic distance, different living habitats or Pleistocene climatic oscillations have frequently been found to shape population genetic structure in many species. The genetic structure of Schizothorax nukiangensis, a high altitude, valuable fish species, which is distributed throughout the Nujiang River, was investigated by mitochondrial DNA sequence analysis. The cytochrome c oxidase subunit I (COI), cytochrome b (cytb), and the mitochondrial control region (MCR) of S. nukiangensis were concatenated for examination of population structure and demographic history. The concatenated data set (2405 bp) implied a pronounced genetic population structure (overall FST = 0.149) and defined two population units. Strong differentiation was detected between the Sanjiangkou (SJK) population and other populations due to environmental heterogeneity, dispersal ability, and/or glacial cycles. Additional DNA sequencing of the nuclear RAG2 gene also examined significant differentiation between two units and between SJK and the upstream populations (U-unit). Recent expansion events suggest that S. nukiangensis may have undergone a rapid increase during warm interglacial periods. Surprisingly, S. nukiangensis appears to have undergone an obvious expansion during the last glaciations (LG) for cold hardiness and a sharp contraction from 1.5 ka to the present. However, two population units exhibited different reflections during the LG, which might be closely related to their living habitats and cold hardiness. A clear pattern of isolation by distance was detected in S. nukiangensis due to feeding habits, limited dispersal ability, and/or philopatry. It is vitally important that more attention be given to S. nukiangensis due to low genetic diversity, lack of gene flow, and recent population contraction.